SUMMARY

Clinical, epidemiological, and microbiological studies have
conclusively shown that the diagnosis of streptococcal infections based
on clinical symptoms always require microbiological verification.(4)
Beta-hemolytic streptococci are the most frequently isolated human pathogens among the representatives of the genus
Streptococcus.
Nearly all the beta-hemolytic streptococci possess specific
carbohydrate antigens, known as streptococcal grouping antigens.
Previously Lancefield showed that these antigens can be extracted in
soluble form and identified by precipitation reactions with homologous
antisera. Currently different procedures for extraction of
streptococcal antigens are in use.(1,2,6,7)

Hardy Diagnostics StrepPRO™ Grouping Kit liberates the
specific antigen from the bacterial cell wall using a modified nitrous
acid extraction. The extracted antigen, in conjunction with latex
agglutination, offers a rapid, sensitive, and specific method for
identification of streptococcal groups A, B, C, D, F, and G from
primary culture plates. Extraction Reagents 1 and 2 contain a chemical
substance able to extract the streptococcal group specific antigens at
room temperature. Extraction Reagent 3 contains a neutralizing
solution. The neutralized extracts can then be easily identified using
blue latex particles sensitized with purified group specific rabbit
immunoglobulins. These blue latex particles agglutinate strongly in the
presence of homologous antigen and will not agglutinate when homologous
antigen is absent.

MATERIALS SUPPLIED

Blue Latex Suspension Group A

Blue
latex particles coated with purified rabbit antibodies to group A
streptococci, suspended in phosphate buffer with 0.1% sodium azide

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops,
pasteur pipettes, 12x75mm test tube, timers, other culture media,
swabs, applicator sticks, incinerators, and incubators, etc., as well
as serological and biochemical reagents, are not provided.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8°C away from direct light.
Do not freeze.
This kit, or any of its reagents, should not be used if there are any
signs of discoloration, contamination, or if the expiration date has
passed.

PRECAUTIONS

The Blue Latex Reagents, Extraction Reagent 1, and Extraction
Reagent 3 contain sodium azide as a preservative. Sodium azide can
react explosively with copper or lead if allowed to accumulate.
Although the amount of sodium azide in the reagents is minimal, large
quantities of water should be used when flushing these reagents down
the sink.

Extraction Reagents 1, 2, and 3 contain a caustic agent. In case of
skin contact, immediately wash with soap and copious amounts of water.
In case of eye contact, flush for at least 15 minutes with water.

PROCEDURE

In general, an overnight, gram-positive, beta-hemolytic isolate of
streptococcal isolate is required for use in this assay. Colonies
should be chosen from an area demonstrating obvious isolation. One to
four isolated colonies are recommended for grouping testing, however,
if colonies are pinpoint, an increased number of colonies,
approximately a loopful, should be used.

Test Protocol:

1. Allow all reagents to acclimatize to room temperature for 10 minutes prior to use.

2. Label one 12x75mm test tube for each specimen tested.

3. Add one drop of Extraction Reagent 1 to each tube.

4. Using a loop, select 1 to 4 isolated beta-hemolytic colonies and
suspend them in Extraction Reagent 1. If colonies are minute, pick a
loopful of colonies, such that the Extraction Reagent 1 becomes turbid.

5. Add one drop of Extraction Reagent 2 to each tube.

6. Mix the reaction by tapping the tube with a finger for 10 seconds.

7. Add five drops of Extraction Reagent 3 to each tube. Mix the reaction as in step 6.

8. Prior to use, resuspend the Blue Latex Reagents, by inverting the
tubes. Dispense one drop of each blue latex suspension onto separate
circles on the test card.

9. Using a pasteur pipette, place one drop of the extract suspension
next to the drop of latex suspension in each circle. Ensure that the
pipette tip does not touch the latex suspension.

10. Mix the blue latex and the extract with the wooden sticks
provided, using the complete area of the circle. A new stick should be
used for each reagent.

12. At one minute, under normal lighting conditions, observe for
agglutination, or strong clumping, of the blue latex particles. See
Interpretation of Results for more information.

INTERPRETATION OF RESULTS

Positive Results:
A rapid and significantly strong clumping of the blue latex particles,
to form an agglutination pattern, with only one of the latex reagents
indicates an identification of the streptococcal isolate for the
Lancefield group.

A weak reaction with a single blue latex reagent should be repeated
using a heavier inoculum. The repeated test is considered positive if a
visible agglutination occurs with only one of the blue latex reagents.

Negative Results:
No visible agglutination of the latex particles indicates a negative reaction for the particular Lancefield group.

FLOW CHART

Figure 1: Suggested Scheme for Streptococcal Grouping:

LIMITATIONS

This kit is intended for use in the identification of beta-hemolytic
streptococci. If alpha or gamma streptococci are identified, the
identification should be confirmed by biochemical tests. See flow chart
for the recommended procedure for grouping streptococci.(5,9)

Do not perform the procedure using a broth culture as false reactions can occur.

False-negative or false-positive results can occur if inadequate amounts of culture or Extraction Reagents are used.

False-positive reactions have been known to occur with organisms from unrelated genera, including gram-negatives such as
Escherichia coli, Klebsiella, and Pseudomonas species. These are likely to non-specifically agglutinate all latex
reagents. This kit is recommended only for testing of gram-positive
organisms.

Listeria monocytogenes
may cross react with the Streptococcal Latex Reagents Group B and/or Group G, since
L. monocytogenes
exhibits similar antigenicity to group B and G streptococci. The catalase test may be performed to distinguish between
Listeria,
which are catalase-positive, and streptococci, which are
catalase-negative. Gram staining and motility testing may be performed
as further aids in differentiation.

Some rare strains of group D streptococci have been found to cross
react with group G antisera. These strains may be confirmed as group D
by a positive Bile Esculin (Cat. no. G12 or L10) test.

QUALITY CONTROL

The following organisms are routinely tested on each lot of StrepPRO™ Grouping Kit by the manufacturer:

Test Organisms

Inoculation Method*

Incubation

Results

Time

Temperature

Atmosphere

Streptococcus pyogenes
ATCC® 19615

*

1 min.

35°C

Aerobic or CO 2**

Agglutination observed only with Blue Latex Suspension Group A

Streptococcus agalactiae
(Group B)
ATCC® 12386

*

1 min.

35°C

Aerobic or CO2**

Agglutination observed only with Blue Latex Suspension Group B

Streptococcus
spp.
(Group C)
ATCC® 12388

*

1 min.

35°C

Aerobic or CO2**

Agglutination observed only with Blue Latex Suspension Group C

Enterococcus faecalis
ATCC® 19433

*

1 min.

35°C

Aerobic or CO2**

Agglutination observed only with Blue Latex Suspension Group D

Streptococcus
spp.
(Group F)
ATCC® 12392

*

1 min.

35°C

Aerobic

Agglutination observed only with Blue Latex Suspension Group F

Streptococcus
spp.
(Group G)
ATCC® 12394

*

1 min.

35°C

Aerobic

Agglutination observed only with Blue Latex Suspension Group G

In addition, each Blue Latex Suspension is tested for the absence of cross-reactions against extracts of the following
additional quality control organisms:

Test Organisms

Inoculation Method*

Incubation

Results

Time

Temperature

Atmosphere

Escherichia coli
ATCC® 25922

*

1 min.

35°C

Aerobic or CO2**

No agglutination observed with any Blue Latex Suspensions

Klebsiella pneumoniae
ATCC® 13883

*

1 min.

35°C

Aerobic or CO2**

No agglutination observed with any Blue Latex Suspensions

Staphylococcus aureus
ATCC® 25923

*

1 min.

35°C

Aerobic or CO2**

No agglutination observed with any Blue Latex Suspensions

Haemophilus influenza
Type B
ATCC® 10211

*

1 min.

35°C

Aerobic or CO2**

No agglutination observed with any Blue Latex Suspensions

* Refer to the section entitled Procedure for a detailed description of the inoculation protocol.

END USER QUALITY CONTROL

Latex Suspension
Verify the performance of each Latex Suspension by agglutinating with the Polyvalent Positive Control (included in the kit).
Expected Results: positive agglutination.

Extraction Reagents
Verify the performance of Extraction Reagents 1, 2, and 3 with
S. pyogenes ATCC® 19615. Agglutinate the extract with the Latex Suspension Group A that was previously approved in the step above.
Expected Results: positive agglutination.

Absence of Autoagglutination
Verify the lack of autoagglutination by performing the test procedure
on one of the Latex Reagents without adding bacteria or the Polyvalent
Positive Control to the Extraction Reagents.
Expected Results: negative agglutination.

Hardy Diagnostics StrepPRO™ Grouping Kit was evaluated at a
Microbiological Center in Oxford, England. In this study 468 primary
culture were tested by the StrepPRO™ Grouping Kit and an
alternative grouping kit. Overall agreement between the two kits upon
first time testing occurred with 452 of 468 isolates tested (96.6%).
Anomalous results (N=16; all minute colonies) were repeated using a
heavier inoculum. 13 of the 16 anomalous results agreed after retest
which included 1 group A, 2 group B, 3 group D, 1 group F, 5 group G,
and 1 non-groupable strains. Two of the 3 discrepant strains were
further identified as non-beta-hemolytic strains, while the third
isolate grouped as group D by StrepPRO™ Grouping Kit, while
determined non-groupable by the alternative kit. This isolate gave a
positive group D isolate result with the alternative kit following
subculture. Overall agreement between the StrepPRO™ Grouping Kit
and the alternative grouping kit after retest of anomalous results
occurred with 463 of 468 isolates tested (99.4%). The 468 Streptococci
isolates used in this study included 127 group A, 93 group B, 30 group
C, 28 group D, 8 group F, 107 group G, and 75 non-groupable strains.

A second performance study was carried out a Health Center in
Ontario, Canada. In this study, 111 primary cultures were included (110
tested, 1 inadequate). All the strains were originally grouped by
Lancefield precipitation reactions. All group D were further
biochemically confirmed using Bile Esculin and PYR assay protocol. The
primary cultures were tested in parallel using the StrepPRO™
Grouping Kit and an alternative grouping kit. In this study, the
overall agreement between StrepPRO™ Grouping Kit and the
Lancefield results occurred with 109 of the 110 isolates tested (99%),
while overall agreement between the alternative kit and Lancefield
results occurred with 106 of 110 isolates tested (96.3%). The 110
Streptococci primary isolates used in this study included 15 group A,
40 group B, 13 group C, 4 group D, 11 group F, 12 group G, and 15
non-groupable strains.